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1.
Cell ; 182(5): 1341-1359.e19, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32763154

RESUMO

Antitumoral immunity requires organized, spatially nuanced interactions between components of the immune tumor microenvironment (iTME). Understanding this coordinated behavior in effective versus ineffective tumor control will advance immunotherapies. We re-engineered co-detection by indexing (CODEX) for paraffin-embedded tissue microarrays, enabling simultaneous profiling of 140 tissue regions from 35 advanced-stage colorectal cancer (CRC) patients with 56 protein markers. We identified nine conserved, distinct cellular neighborhoods (CNs)-a collection of components characteristic of the CRC iTME. Enrichment of PD-1+CD4+ T cells only within a granulocyte CN positively correlated with survival in a high-risk patient subset. Coupling of tumor and immune CNs, fragmentation of T cell and macrophage CNs, and disruption of inter-CN communication was associated with inferior outcomes. This study provides a framework for interrogating how complex biological processes, such as antitumoral immunity, occur through concerted actions of cells and spatial domains.


Assuntos
Neoplasias Colorretais/imunologia , Neoplasias Colorretais/terapia , Invasividade Neoplásica/imunologia , Antígeno B7-H1/imunologia , Biomarcadores Tumorais/imunologia , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular Tumoral , Feminino , Humanos , Imunoterapia/métodos , Masculino , Microambiente Tumoral/imunologia
2.
Cell ; 183(5): 1383-1401.e19, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33159858

RESUMO

Ebola virus (EBOV) causes epidemics with high mortality yet remains understudied due to the challenge of experimentation in high-containment and outbreak settings. Here, we used single-cell transcriptomics and CyTOF-based single-cell protein quantification to characterize peripheral immune cells during EBOV infection in rhesus monkeys. We obtained 100,000 transcriptomes and 15,000,000 protein profiles, finding that immature, proliferative monocyte-lineage cells with reduced antigen-presentation capacity replace conventional monocyte subsets, while lymphocytes upregulate apoptosis genes and decline in abundance. By quantifying intracellular viral RNA, we identify molecular determinants of tropism among circulating immune cells and examine temporal dynamics in viral and host gene expression. Within infected cells, EBOV downregulates STAT1 mRNA and interferon signaling, and it upregulates putative pro-viral genes (e.g., DYNLL1 and HSPA5), nominating pathways the virus manipulates for its replication. This study sheds light on EBOV tropism, replication dynamics, and elicited immune response and provides a framework for characterizing host-virus interactions under maximum containment.


Assuntos
Ebolavirus/fisiologia , Doença pelo Vírus Ebola/genética , Doença pelo Vírus Ebola/virologia , Interações Hospedeiro-Patógeno/genética , Análise de Célula Única , Animais , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Efeito Espectador , Diferenciação Celular , Proliferação de Células , Citocinas/metabolismo , Ebolavirus/genética , Chaperona BiP do Retículo Endoplasmático , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Regulação Viral da Expressão Gênica , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/patologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Interferons/genética , Interferons/metabolismo , Macaca mulatta , Macrófagos/metabolismo , Monócitos/metabolismo , Mielopoese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Transcriptoma/genética
3.
Immunity ; 55(6): 1118-1134.e8, 2022 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-35447093

RESUMO

Understanding the mechanisms of HIV tissue persistence necessitates the ability to visualize tissue microenvironments where infected cells reside; however, technological barriers limit our ability to dissect the cellular components of these HIV reservoirs. Here, we developed protein and nucleic acid in situ imaging (PANINI) to simultaneously quantify DNA, RNA, and protein levels within these tissue compartments. By coupling PANINI with multiplexed ion beam imaging (MIBI), we measured over 30 parameters simultaneously across archival lymphoid tissues from healthy or simian immunodeficiency virus (SIV)-infected nonhuman primates. PANINI enabled the spatial dissection of cellular phenotypes, functional markers, and viral events resulting from infection. SIV infection induced IL-10 expression in lymphoid B cells, which correlated with local macrophage M2 polarization. This highlights a potential viral mechanism for conditioning an immunosuppressive tissue environment for virion production. The spatial multimodal framework here can be extended to decipher tissue responses in other infectious diseases and tumor biology.


Assuntos
Infecções por HIV , Ácidos Nucleicos , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Animais , Linfócitos T CD4-Positivos , Vírus de DNA , Terapia de Imunossupressão , Macaca mulatta , Macrófagos , Vírus da Imunodeficiência Símia/fisiologia , Carga Viral
5.
Nat Methods ; 20(2): 304-315, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36624212

RESUMO

The ability to align individual cellular information from multiple experimental sources is fundamental for a systems-level understanding of biological processes. However, currently available tools are mainly designed for single-cell transcriptomics matching and integration, and generally rely on a large number of shared features across datasets for cell matching. This approach underperforms when applied to single-cell proteomic datasets due to the limited number of parameters simultaneously accessed and lack of shared markers across these experiments. Here, we introduce a cell-matching algorithm, matching with partial overlap (MARIO) that accounts for both shared and distinct features, while consisting of vital filtering steps to avoid suboptimal matching. MARIO accurately matches and integrates data from different single-cell proteomic and multimodal methods, including spatial techniques and has cross-species capabilities. MARIO robustly matched tissue macrophages identified from COVID-19 lung autopsies via codetection by indexing imaging to macrophages recovered from COVID-19 bronchoalveolar lavage fluid by cellular indexing of transcriptomes and epitopes by sequencing, revealing unique immune responses within the lung microenvironment of patients with COVID.


Assuntos
COVID-19 , Proteômica , Humanos , Proteômica/métodos , Perfilação da Expressão Gênica/métodos , Transcriptoma , Pulmão , Análise de Célula Única/métodos
6.
Proc Natl Acad Sci U S A ; 119(6)2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35110410

RESUMO

Despite more than 300,000 rVSVΔG-ZEBOV-glycoprotein (GP) vaccine doses having been administered during Ebola virus disease (EVD) outbreaks in the Democratic Republic of the Congo (DRC) between 2018 and 2020, seroepidemiologic studies of vaccinated Congolese populations are lacking. This study examines the antibody response at 21 d and 6 mo postvaccination after single-dose rVSVΔG-ZEBOV-GP vaccination among EVD-exposed and potentially exposed populations in the DRC. We conducted a longitudinal cohort study of 608 rVSVΔG-ZEBOV-GP-vaccinated individuals during an EVD outbreak in North Kivu Province, DRC. Participants provided questionnaires and blood samples at three study visits (day 0, visit 1; day 21, visit 2; and month 6, visit 3). Anti-GP immunoglobulin G (IgG) antibody titers were measured in serum by the Filovirus Animal Nonclinical Group anti-Ebola virus GP IgG enzyme-linked immunosorbent assay. Antibody response was defined as an antibody titer that had increased fourfold from visit 1 to visit 2 and was above four times the lower limit of quantification at visit 2; antibody persistence was defined as a similar increase from visit 1 to visit 3. We then examined demographics for associations with follow-up antibody titers using generalized linear mixed models. A majority of the sample, 87.2%, had an antibody response at visit 2, and 95.6% demonstrated antibody persistence at visit 3. Being female and of young age was predictive of a higher antibody titer postvaccination. Antibody response and persistence after Ebola vaccination was robust in this cohort, confirming findings from outside of the DRC.


Assuntos
Vacinas contra Ebola/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Imunogenicidade da Vacina/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/imunologia , Criança , República Democrática do Congo , Surtos de Doenças/prevenção & controle , Feminino , Glicoproteínas/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Soroepidemiológicos , Vacinação/métodos , Proteínas do Envelope Viral/imunologia , Adulto Jovem
7.
PLoS Pathog ; 18(9): e1010802, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36095030

RESUMO

The impact of vaccination on SARS-CoV-2 infectiousness is not well understood. We compared longitudinal viral shedding dynamics in unvaccinated and fully vaccinated adults. SARS-CoV-2-infected adults were enrolled within 5 days of symptom onset and nasal specimens were self-collected daily for two weeks and intermittently for an additional two weeks. SARS-CoV-2 RNA load and infectious virus were analyzed relative to symptom onset stratified by vaccination status. We tested 1080 nasal specimens from 52 unvaccinated adults enrolled in the pre-Delta period and 32 fully vaccinated adults with predominantly Delta infections. While we observed no differences by vaccination status in maximum RNA levels, maximum infectious titers and the median duration of viral RNA shedding, the rate of decay from the maximum RNA load was faster among vaccinated; maximum infectious titers and maximum RNA levels were highly correlated. Furthermore, amongst participants with infectious virus, median duration of infectious virus detection was reduced from 7.5 days (IQR: 6.0-9.0) in unvaccinated participants to 6 days (IQR: 5.0-8.0) in those vaccinated (P = 0.02). Accordingly, the odds of shedding infectious virus from days 6 to 12 post-onset were lower among vaccinated participants than unvaccinated participants (OR 0.42 95% CI 0.19-0.89). These results indicate that vaccination had reduced the probability of shedding infectious virus after 5 days from symptom onset.


Assuntos
COVID-19 , SARS-CoV-2 , Adulto , COVID-19/prevenção & controle , Humanos , Estudos Longitudinais , RNA Viral/genética , Vacinação , Eliminação de Partículas Virais
8.
J Biol Chem ; 295(13): 4350-4358, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32060096

RESUMO

The metalloprotease ADAM17 (a disintegrin and metalloprotease 17) is a key regulator of tumor necrosis factor α (TNFα), interleukin 6 receptor (IL-6R), and epidermal growth factor receptor (EGFR) signaling. ADAM17 maturation and function depend on the seven-membrane-spanning inactive rhomboid-like proteins 1 and 2 (iRhom1/2 or Rhbdf1/2). Most studies to date have focused on overexpressed iRhom1 and -2, so only little is known about the properties of the endogenous proteins. Here, we show that endogenous iRhom1 and -2 can be cell surface-biotinylated on mouse embryonic fibroblasts (mEFs), revealing that endogenous iRhom1 and -2 proteins are present on the cell surface and that iRhom2 also is present on the surface of lipopolysaccharide-stimulated primary bone marrow-derived macrophages. Interestingly, very little, if any, iRhom2 was detectable in mEFs or bone marrow-derived macrophages lacking ADAM17, suggesting that iRhom2 is stabilized by ADAM17. By contrast, the levels of iRhom1 were slightly increased in the absence of ADAM17 in mEFs, indicating that its stability does not depend on ADAM17. These findings support a model in which iRhom2 and ADAM17 are obligate binding partners and indicate that iRhom2 stability requires the presence of ADAM17, whereas iRhom1 is stable in the absence of ADAM17.


Assuntos
Proteína ADAM17/genética , Proteínas de Transporte/genética , Proteínas de Membrana/genética , Fator de Necrose Tumoral alfa/genética , Animais , Membrana Celular , Receptores ErbB/genética , Fibroblastos/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Camundongos , Receptores de Interleucina-6/genética , Transdução de Sinais/genética
9.
FASEB J ; 34(4): 4956-4969, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32103528

RESUMO

The metalloprotease ADAM17 (a disintegrin and metalloprotease 17) regulates EGF-receptor and TNFα signaling, thereby not only protecting the skin and intestinal barrier, but also contributing to autoimmunity. ADAM17 can be rapidly activated by many stimuli through its transmembrane domain (TMD), with the seven membrane-spanning inactive Rhomboids (iRhom) 1 and 2 implicated as candidate regulatory partners. However, several alternative models of ADAM17 regulation exist that do not involve the iRhoms, such as regulation through disulfide bond exchange or through interaction with charged phospholipids. Here, we report that a non-activatable mutant of ADAM17 with the TMD of betacellulin (BTC) can be rescued by restoring residues from the ADAM17 TMD, but only in Adam17-/- cells, which contain iRhoms, not in iRhom1/2-/- cells. We also provide the first evidence that the extracellular juxtamembrane domains (JMDs) of ADAM17 and iRhom2 regulate the stimulation and substrate selectivity of ADAM17. Interestingly, a point mutation in the ADAM17 JMD identified in a patient with Tetralogy of Fallot, a serious heart valve defect, affects the substrate selectivity of ADAM17 toward Heparin-binding epidermal growth factor like growth factor (HB-EGF), a crucial regulator of heart valve development in mice. These findings provide new insights into the regulation of ADAM17 through an essential interaction with the TMD1 and JMD1 of iRhom2.


Assuntos
Proteína ADAM17/metabolismo , Proteínas de Transporte/metabolismo , Mutação , Proteína ADAM17/química , Proteína ADAM17/genética , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Domínios Proteicos , Especificidade por Substrato , Tetralogia de Fallot/genética
10.
Virol J ; 18(1): 45, 2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33632249

RESUMO

BACKGROUND: Influenza places a significant burden on global health and economics. Individual case management and public health efforts to mitigate the spread of influenza are both strongly impacted by our ability to accurately and efficiently detect influenza viruses in clinical samples. Therefore, it is important to understand the performance characteristics of available assays to detect influenza in a variety of settings. We provide the first report of relative performance between two products marketed to streamline detection of influenza virus in the context of a highly controlled volunteer influenza challenge study. METHODS: Nasopharyngeal swab samples were collected during a controlled A/California/2009/H1N1 influenza challenge study and analyzed for detection of virus shedding using a validated qRT-PCR (qPCR) assay, a sample-to-answer qRT-PCR device (BioMerieux BioFire FilmArray RP), and an immunoassay based rapid test kit (Quidel QuickVue Influenza A + B Test). RESULTS: Relative to qPCR, the sensitivity and specificity of the BioFire assay was 72.1% [63.7-79.5%, 95% confidence interval (CI)] and 93.5% (89.3-96.4%, 95% CI) respectively. For the QuickVue rapid test the sensitivity was 8.5% (4.8-13.7%, 95% CI) and specificity was 99.2% (95.6-100%, 95% CI). CONCLUSION: Relative to qPCR, the BioFire assay had superior performance compared to rapid test in the context of a controlled influenza challenge study.


Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza B/genética , Influenza Humana/diagnóstico , Técnicas de Diagnóstico Molecular/normas , Reação em Cadeia da Polimerase em Tempo Real/normas , Experimentação Humana , Humanos , Influenza Humana/virologia , Técnicas de Diagnóstico Molecular/métodos , Nasofaringe/virologia , Kit de Reagentes para Diagnóstico/normas , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Eliminação de Partículas Virais , Voluntários
11.
BMC Immunol ; 21(1): 15, 2020 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-32228458

RESUMO

BACKGROUND: Cryopreserved human peripheral blood mononuclear cells (PBMCs) are a commonly used sample type for a variety of immunological assays. Many factors can affect the quality of PBMCs, and careful consideration and validation of an appropriate PBMC isolation and cryopreservation method is important for well-designed clinical studies. A major point of divergence in PBMC isolation protocols is the collection of blood, either directly into vacutainers pre-filled with density gradient medium or the use of conical tubes containing a porous barrier to separate the density gradient medium from blood. To address potential differences in sample outcome, we isolated, cryopreserved, and compared PBMCs using parallel protocols differing only in the use of one of two common tube types for isolation. METHODS: Whole blood was processed in parallel using both Cell Preparation Tubes™ (CPT, BD Biosciences) and Lymphoprep™ Tubes (Axis-Shield) and assessed for yield and viability prior to cryopreservation. After thawing, samples were further examined by flow cytometry for cell yield, cell viability, frequency of 10 cell subsets, and capacity for stimulation-dependent CD4+ and CD8+ T cell intracellular cytokine production. RESULTS: No significant differences in cell recovery, viability, frequency of immune cell subsets, or T cell functionality between PBMC samples isolated using CPT or Lymphoprep tubes were identified. CONCLUSION: CPT and Lymphoprep tubes are effective and comparable methods for PBMC isolation for immunological studies.


Assuntos
Separação Celular/métodos , Criopreservação/métodos , Ficoll/química , Leucócitos Mononucleares/citologia , Ácido Metrizoico/química , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Sobrevivência Celular/imunologia , Células Cultivadas , Citocinas/imunologia , Citometria de Fluxo/métodos , Humanos , Leucócitos Mononucleares/imunologia
12.
Bioinformatics ; 35(1): 95-103, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30561547

RESUMO

Motivation: Multiple biological clocks govern a healthy pregnancy. These biological mechanisms produce immunologic, metabolomic, proteomic, genomic and microbiomic adaptations during the course of pregnancy. Modeling the chronology of these adaptations during full-term pregnancy provides the frameworks for future studies examining deviations implicated in pregnancy-related pathologies including preterm birth and preeclampsia. Results: We performed a multiomics analysis of 51 samples from 17 pregnant women, delivering at term. The datasets included measurements from the immunome, transcriptome, microbiome, proteome and metabolome of samples obtained simultaneously from the same patients. Multivariate predictive modeling using the Elastic Net (EN) algorithm was used to measure the ability of each dataset to predict gestational age. Using stacked generalization, these datasets were combined into a single model. This model not only significantly increased predictive power by combining all datasets, but also revealed novel interactions between different biological modalities. Future work includes expansion of the cohort to preterm-enriched populations and in vivo analysis of immune-modulating interventions based on the mechanisms identified. Availability and implementation: Datasets and scripts for reproduction of results are available through: https://nalab.stanford.edu/multiomics-pregnancy/. Supplementary information: Supplementary data are available at Bioinformatics online.


Assuntos
Metaboloma , Microbiota , Gravidez , Proteoma , Transcriptoma , Biologia Computacional , Feminino , Humanos
13.
Blood ; 132(10): 1064-1074, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-29776906

RESUMO

Hemophilic arthropathy (HA) is a debilitating degenerative joint disease that is a major manifestation of the bleeding disorder hemophilia A. HA typically begins with hemophilic synovitis that resembles inflammatory arthritides, such as rheumatoid arthritis, and frequently results in bone loss in patients. A major cause of rheumatoid arthritis is inappropriate release of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) by the TNF-α convertase (TACE; also referred to as ADAM17) and its regulator, iRhom2. Therefore, we hypothesized that iRhom2/ADAM17-dependent shedding of TNF-α also has a pivotal role in mediating HA. Here, we show that addition of blood or its components to macrophages activates iRhom2/ADAM17-dependent TNF-α shedding, providing the premise to study the activation of this pathway by blood in the joint in vivo. For this, we turned to hemophilic FVIII-deficient mice (F8-/- mice), which develop a hemarthrosis following needle puncture injury with synovial inflammation and significant osteopenia adjacent to the affected joint. We found that needle puncture-induced bleeding leads to increased TNF-α levels in the affected joint of F8-/- mice. Moreover, inactivation of TNF-α or iRhom2 in F8-/- mice reduced the osteopenia and synovial inflammation that develops in this mouse model for HA. Taken together, our results suggest that blood entering the joint activates the iRhom2/ADAM17/TNF-α pathway, thereby contributing to osteopenia and synovitis in mice. Therefore, this proinflammatory signaling pathway could emerge as an attractive new target to prevent osteoporosis and joint damage in HA patients.


Assuntos
Proteína ADAM17/metabolismo , Reabsorção Óssea/metabolismo , Proteínas de Transporte/metabolismo , Hemartrose/metabolismo , Hemofilia A/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Proteína ADAM17/genética , Animais , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Proteínas de Transporte/genética , Modelos Animais de Doenças , Fator VIII/genética , Feminino , Hemartrose/genética , Hemartrose/patologia , Hemofilia A/genética , Hemofilia A/patologia , Camundongos , Camundongos Knockout , Fator de Necrose Tumoral alfa/genética
14.
Int J Mol Sci ; 21(22)2020 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-33227998

RESUMO

Growth of the axial and appendicular skeleton depends on endochondral ossification, which is controlled by tightly regulated cell-cell interactions in the developing growth plates. Previous studies have uncovered an important role of a disintegrin and metalloprotease 17 (ADAM17) in the normal development of the mineralized zone of hypertrophic chondrocytes during endochondral ossification. ADAM17 regulates EGF-receptor signaling by cleaving EGFR-ligands such as TGFα from their membrane-anchored precursor. The activity of ADAM17 is controlled by two regulatory binding partners, the inactive Rhomboids 1 and 2 (iRhom1, 2), raising questions about their role in endochondral ossification. To address this question, we generated mice lacking iRhom2 (iR2-/-) with floxed alleles of iRhom1 that were specifically deleted in chondrocytes by Col2a1-Cre (iR1∆Ch). The resulting iR2-/-iR1∆Ch mice had retarded bone growth compared to iR2-/- mice, caused by a significantly expanded zone of hypertrophic mineralizing chondrocytes in the growth plate. Primary iR2-/-iR1∆Ch chondrocytes had strongly reduced shedding of TGFα and other ADAM17-dependent EGFR-ligands. The enlarged zone of mineralized hypertrophic chondrocytes in iR2-/-iR1∆Ch mice closely resembled the abnormal growth plate in A17∆Ch mice and was similar to growth plates in Tgfα-/- mice or mice with EGFR mutations. These data support a model in which iRhom1 and 2 regulate bone growth by controlling the ADAM17/TGFα/EGFR signaling axis during endochondral ossification.


Assuntos
Proteína ADAM17/genética , Proteínas de Transporte/genética , Condrócitos/metabolismo , Condrogênese/genética , Proteínas de Membrana/genética , Osteogênese/genética , Proteína ADAM17/metabolismo , Animais , Calcificação Fisiológica/genética , Proteínas de Transporte/metabolismo , Comunicação Celular , Diferenciação Celular , Proliferação de Células , Condrócitos/citologia , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação da Expressão Gênica , Lâmina de Crescimento/crescimento & desenvolvimento , Lâmina de Crescimento/metabolismo , Integrases/genética , Integrases/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Transdução de Sinais , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador alfa/metabolismo
15.
Clin Infect Dis ; 68(8): 1388-1393, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30107392

RESUMO

BACKGROUND: Clinical sequelae of Ebola virus disease (EVD) have not been described more than 3 years postoutbreak. We examined survivors and close contacts from the 1995 Ebola outbreak in Kikwit, Democratic Republic of Congo (DRC), and determined prevalence of abnormal neurological, cognitive, and psychological findings and their association with EVD survivorship. METHODS: From August to September 2017, we conducted a cross-sectional study in Kikwit, DRC. Over 2 decades after the EVD outbreak, we recruited EVD survivors and close contacts from the outbreak to undergo physical examination and culturally adapted versions of the Folstein mini-mental status exam (MMSE) and Goldberg anxiety and depression scale (GADS). We estimated the strength of relationships between EVD survivorship and health outcomes using linear regression models by comparing survivors versus close contacts, adjusting for age, sex, educational level, marital status, and healthcare worker status. RESULTS: We enrolled 20 EVD survivors and 187 close contacts. Among the 20 EVD survivors, 4 (20%) reported at least 1 abnormal neurological symptom, and 3 (15%) had an abnormal neurological examination. Among the 187 close contacts, 14 (11%) reported at least 1 abnormal neurologic symptom, and 9 (5%) had an abnormal neurological examination. EVD survivors had lower mean MMSE and higher mean GADS scores as compared to close contacts (MMSE: adjusted coefficient: -1.85; 95% confidence interval [CI]: -3.63, -0.07; GADS: adjusted coefficient: 3.91; 95% CI: 1.76, 6.04). CONCLUSIONS: EVD survivors can have lower cognitive scores and more symptoms of depression and anxiety than close contacts more than 2 decades after Ebola virus outbreaks.


Assuntos
Doença pelo Vírus Ebola/fisiopatologia , Doença pelo Vírus Ebola/psicologia , Ansiedade , Cognição , Estudos Transversais , República Democrática do Congo/epidemiologia , Depressão , Surtos de Doenças , Feminino , Doença pelo Vírus Ebola/epidemiologia , Humanos , Masculino , Testes de Estado Mental e Demência , Pessoa de Meia-Idade , Sobreviventes
16.
J Cell Physiol ; 233(3): 2247-2256, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28703301

RESUMO

The xenoestrogens bisphenol-A (BPA) and nonylphenol (NP) are endocrine disruptors used in the plastic polymer industry to manufacture different products for human use. Previous studies have suggested a role of these compounds in the shedding of signaling molecules, such as tumor necrosis factor α (TNF-α). The aim of this work was to evaluate the effect of BPA and NP on the sheddase ADAM17 and its newly discovered regulators iRhom1 and iRhom2 in the release of EGFR-ligands. We report that BPA and NP can stimulate the release of the ADAM17-substrates HB-EGF and TGF-α. In cells lacking ADAM17 (Adam17-/- mEFs) BPA-stimulated release of HB-EGF, but not TGF-α, was strongly reduced, whereas NP-stimulated shedding of HB-EGF and TGF-α was completely abolished. Inactivation of both ADAM17 and the related ADAM10 (Adam10/17-/- mEFs) completely prevented the release of these substrates. In the absence of iRhom1, BPA- or NP-stimulated release of HB-EGF or TGF-α was comparable to wild-type control mEFs, conversely the BPA-induced release of HB-EGF was abolished in iRhom2-/- mEFs. The defect in shedding of HB-EGF in iRhom2-/- mEF cells could be rescued by overexpressing iRhom2. Interestingly, the NP-stimulated release of HB-EGF was not affected by the absence of iRhom2, suggesting that NP could potentially activate both ADAM10 and ADAM17. We tested this hypothesis using betacellulin (BTC), an EGFR-ligand that is a substrate for ADAM10. We found that NP, but not BPA stimulated the release of BTC in Adam17-/- , iRhom2-/- , or iRhom1/2-/- , but not in Adam10/17-/- cells. Taken together, our results suggest that BPA and NP stimulate the release of EGFR-ligands by differentially activating ADAM17 or ADAM10. The identification of specific effects of these endocrine disruptors on ADAM10 and ADAM17 will help to provide a better understanding of their roles in cell signaling and proinflammatory processes, and provide new potential targets for treatment of reproductive or inflammatory diseases such as asthma or breast cancer that are promoted by xenoestrogens.


Assuntos
Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Compostos Benzidrílicos/farmacologia , Disruptores Endócrinos/farmacologia , Receptores ErbB/metabolismo , Estrogênios/farmacologia , Fibroblastos/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Fenóis/farmacologia , Proteína ADAM10/genética , Proteína ADAM17/genética , Secretases da Proteína Precursora do Amiloide/genética , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Ativação Enzimática , Fibroblastos/enzimologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Ligantes , Proteínas de Membrana/genética , Camundongos Knockout , Transfecção , Fator de Necrose Tumoral alfa/metabolismo
17.
Proc Natl Acad Sci U S A ; 112(19): 6080-5, 2015 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-25918388

RESUMO

The metalloproteinase ADAM17 (a disintegrin and metalloprotease 17) controls EGF receptor (EGFR) signaling by liberating EGFR ligands from their membrane anchor. Consequently, a patient lacking ADAM17 has skin and intestinal barrier defects that are likely caused by lack of EGFR signaling, and Adam17(-/-) mice die perinatally with open eyes, like Egfr(-/-) mice. A hallmark feature of ADAM17-dependent EGFR ligand shedding is that it can be rapidly and posttranslationally activated in a manner that requires its transmembrane domain but not its cytoplasmic domain. This suggests that ADAM17 is regulated by other integral membrane proteins, although much remains to be learned about the underlying mechanism. Recently, inactive Rhomboid 2 (iRhom2), which has seven transmembrane domains, emerged as a molecule that controls the maturation and function of ADAM17 in myeloid cells. However, iRhom2(-/-) mice appear normal, raising questions about how ADAM17 is regulated in other tissues. Here we report that iRhom1/2(-/-) double knockout mice resemble Adam17(-/-) and Egfr(-/-) mice in that they die perinatally with open eyes, misshapen heart valves, and growth plate defects. Mechanistically, we show lack of mature ADAM17 and strongly reduced EGFR phosphorylation in iRhom1/2(-/-) tissues. Finally, we demonstrate that iRhom1 is not essential for mouse development but regulates ADAM17 maturation in the brain, except in microglia, where ADAM17 is controlled by iRhom2. These results provide genetic, cell biological, and biochemical evidence that a principal function of iRhoms1/2 during mouse development is to regulate ADAM17-dependent EGFR signaling, suggesting that iRhoms1/2 could emerge as novel targets for treatment of ADAM17/EGFR-dependent pathologies.


Assuntos
Proteínas ADAM/metabolismo , Proteínas de Transporte/metabolismo , Receptores ErbB/metabolismo , Proteína ADAM17 , Animais , Separação Celular , Células-Tronco Embrionárias/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Fibroblastos/metabolismo , Citometria de Fluxo , Heterozigoto , Selectina L/metabolismo , Leucócitos/metabolismo , Ligantes , Masculino , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microglia/metabolismo , Neoplasias/metabolismo , Fenótipo , Fosforilação , Regiões Promotoras Genéticas , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
18.
Eur J Immunol ; 46(12): 2737-2748, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27601030

RESUMO

CSF1R (colony stimulating factor 1 receptor) is the main receptor for CSF1 and has crucial roles in regulating myelopoeisis. CSF1R can be proteolytically released from the cell surface by ADAM17 (A disintegrin and metalloprotease 17). Here, we identified CSF1R as a major substrate of ADAM17 in an unbiased degradomics screen. We explored the impact of CSF1R shedding by ADAM17 and its upstream regulator, inactive rhomboid protein 2 (iRhom2, gene name Rhbdf2), on homeostatic development of mouse myeloid cells. In iRhom2-/- mice, we found constitutive accumulation of membrane-bound CSF1R on myeloid cells at steady state, although cell numbers of these populations were not altered. However, in the context of mixed bone marrow (BM) chimera, under competitive pressure, iRhom2-/- BM progenitor-derived monocytes, tissue macrophages and lung DCs showed a repopulation advantage over those derived from wild-type (WT) BM progenitors, suggesting enhanced CSF1R signaling in the absence of iRhom2. In vitro experiments indicate that iRhom2-/- Lin- SCA-1+ c-Kit+ (LSKs) cells, but not granulocyte-macrophage progenitors (GMPs), had faster growth rates than WT cells in response to CSF1. Our results shed light on an important role of iRhom2/ADAM17 pathway in regulation of CSF1R shedding and repopulation of monocytes, macrophages and DCs.


Assuntos
Proteína ADAM17/metabolismo , Células da Medula Óssea/fisiologia , Proteínas de Transporte/metabolismo , Células Progenitoras Mieloides/fisiologia , Mielopoese , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Proteína ADAM17/genética , Animais , Proteínas de Transporte/genética , Células Cultivadas , Células Dendríticas/fisiologia , Feminino , Regulação da Expressão Gênica , Pulmão/patologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Transdução de Sinais , Quimeras de Transplante
19.
Prostate ; 76(8): 722-34, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27088546

RESUMO

PURPOSE: Both prostate cancer and benign prostatic hyperplasia are associated with inflammatory microenvironments. Inflammation is damaging to tissues, but it is unclear how the inflammatory microenvironment protects specialized epithelial cells that function to proliferate and repair the tissue. The objective of this study is to characterize the cell death and cell survival response of the prostatic epithelium in response to inflammation. METHODS: We assessed induction of cell death (TNF, TRAIL, TWEAK, FasL) and cell survival factors (IGFs, hedgehogs, IL-6, FGFs, and TGFs) in inflamed and control mouse prostates by ELISA. Cell death mechanisms were determined by immunoblotting and immunofluorescence for cleavage of caspases and TUNEL. Survival pathway activation was assessed by immunoblotting and immunofluorescence for Mcl-1, Bcl-2, Bcl-XL, and survivin. Autophagy was determined by immunoblotting and immunofluorescence for free and membrane associated light chain 3 (LC-3). RESULTS: Cleavage of all four caspases was significantly increased during the first 2 days of inflammation, and survival protein expression was substantially increased subsequently, maximizing at 3 days. By 5 days of inflammation, 50% of prostatic epithelial cells expressed survivin. Autophagy was also evident during the recovery phase (3 days). Finally, immunofluorescent staining of human specimens indicates strong activation of survival proteins juxtaposed to inflammation in inflamed prostate specimens. CONCLUSIONS: The prostate responds to deleterious inflammation with induction of cell survival mechanisms, most notably survivin and autophagy, demonstrating a coordinated induction of survival factors that protects and expands a specialized set of prostatic epithelial cells as part of the repair and recovery process during inflammation.


Assuntos
Sobrevivência Celular/fisiologia , Inflamação/metabolismo , Próstata/metabolismo , Hiperplasia Prostática/metabolismo , Neoplasias da Próstata/metabolismo , Transdução de Sinais/fisiologia , Microambiente Tumoral/fisiologia , Idoso , Animais , Apoptose/fisiologia , Caspases/metabolismo , Citocina TWEAK , Proteína Ligante Fas/metabolismo , Humanos , Inflamação/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Próstata/patologia , Hiperplasia Prostática/patologia , Neoplasias da Próstata/patologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fatores de Necrose Tumoral/metabolismo
20.
Eur J Immunol ; 45(2): 418-27, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25393615

RESUMO

STAT3 is a critical transcription factor activated downstream of cytokine signaling and is integral for the function of multiple immune cell types. Human mutations in STAT3 cause primary immunodeficiency resulting in impaired control of a variety of infections, including reactivation of latent viruses. In this study, we investigate how T-cell functions of STAT3 contribute to responses to viral infection by inducing chronic lymphocytic choriomeningitis virus (LCMV) infection in mice lacking STAT3 specifically in T cells. Although mice with conditional disruption of STAT3 in T cells were able to mount early responses to viral infection similar to control animals, including expansion of effector T cells, we found generation of T-follicular helper (Tfh) cells to be impaired. As a result, STAT3 T cell deficient mice produced attenuated germinal center reactions, and did not accumulate bone marrow virus specific IgG-secreting cells, resulting in failure to maintain levels of virus-specific IgG or mount neutralizing responses to LCMV in the serum. These effects were associated with reduced control of viral replication and prolonged infection. Our results demonstrate the importance of STAT3 in T cells for the generation of functional long-term humoral immunity to viral infections.


Assuntos
Anticorpos Antivirais/biossíntese , Imunidade Humoral , Imunoglobulina G/biossíntese , Coriomeningite Linfocítica/imunologia , Fator de Transcrição STAT3/imunologia , Linfócitos T Auxiliares-Indutores/patologia , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Linfócitos B/virologia , Doença Crônica , Expressão Gênica , Imunofenotipagem , Coriomeningite Linfocítica/genética , Coriomeningite Linfocítica/patologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Knockout , Fator de Transcrição STAT3/deficiência , Fator de Transcrição STAT3/genética , Transdução de Sinais , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/patologia , Linfócitos T Citotóxicos/virologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/virologia , Replicação Viral
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